Project Summary The N-ethyl-N-nitrosourea (ENU) forward screen in the mouse is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. The applicant will receive rigorous predoctoral training in the cloning and subsequent molecular characterization of ENU alleles. The sponsor?s laboratory recently identified the Dorothy mutation in a screen designed to recover recessive mutations affecting mouse craniofacial and CNS development. Dorothy homozygous embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies. Along with dramatic limb truncations, Dorothy homozygous embryos lack nasal structures derived from the Medial and Lateral Nasal Prominences (MNP and LNP, respectively), resulting in rudimentary and laterally displaced maxillary structures. The laboratory and applicant used exome sequencing to discover the causative genetic malformation to be a missense mutation in Nubp2. NUBP2 is implicated in both the Cytosolic Iron/Sulfur cluster Assembly (CIA) pathway and in the negative regulation of centrosome duplication/ciliogenesis, although nothing is known about the connection between these functions. The craniofacial and limb phenotypes combined with preliminary expression data suggest a role for NUBP2 in the maintenance of a subset of midfacial Cranial Neural Crest Cells (CNCCs), and in the transduction of SHH and/or WNT signaling. The applicant will test the hypothesis that Nubp2 promotes CNCC survival via its role in the SHH and/or WNT pathway. Further experiments will assess NUBP2?s role in the CIA pathway as a possible connection to regulation of centriole duplication and ciliogenesis. Overall, this first study will provide a novel perspective on both ciliopathies and the CIA pathway and demonstrate for the first time the in vivo consequences of mutations in Nubp2. In a complementary study, the applicant will pursue the cloning of the causal variant in another ENU mutation with syndromic malformations. This mutation is heritable but currently uncloned. The applicant will apply both next-generation sequencing as well as standard positional cloning techniques to identify the candidate mutation. Taken together, this support will provide a period of intensive training in forward genetics, molecular embryology and mouse models of human congenital malformation.